| Walnut leaves(Juglans regia L.)are widely distributed in the north china,which were reported to show insecticidal activity.In the current work,we studied the insecticidal activity of ethanol extract from walnut leaves against three agricultural pests,Plutella xylostella L.,Aphis gossypii G.and Pieris rapae L.,explored the mechanisms of theinsecticidal activities of walnut leaves.Taking the phenoloxidase involved in immune defense,as a target,to study the changes of enzyme activity and protein expression during the insecticidal process,and provide a theoretical basis for the targeted killing of walnut leaves.(1)Determination of the insecticidal activity of 50%,70%and 90%ethanol extracts from walnut leaves against Plutella xylostella L.,Aphis gossypii G.and Pieris rapae L.The experiment showed that the 70%ethanol extract had the strongest stomach toxicity,Aphis gossypii G.>Plutella xylostella L.>Pieris rapae L.,among which the medium lethal concentrations were 11.70 mg/mL and 5.07 mg/mL at 30 h and 48 h,respectively.The medium lethal concentrations of Plutella xylostella L.at 3 d and 4 d were 16.92 mg/mL and 8.31 mg/mL,respectively;the intermediate lethal concentrations of Pieris rapae L.at 3 d and 4 d were 21.55 mg/mL and 13.86 mg/mL,respectively.The contact toxicity of 70%ethanol extracts against Aphis gossypii G.was the best,and the insecticidal rate reached 80.15%after 25 mg/mL exposure for 48 h;90%ethanol extracts had the best contact toxicity against Plutella xylostella L.and Pieris rapae L.,the insecticidal rate was 71.23%and 56.12%at the same conditions..The 70%ethanol extracts had the best growth inhibitory effect on Plutella xylostella L.,the body weight inhibition rate reached 34.12%at 24 h.The 90%ethanol extract had the best growth inhibition effect against Pieris rapae L.,the body weight inhibition rate was 41.23%at 24 h.The 90%ethanol extract had the strongest antifeedant activity against the three test species,and the non-selective and selective antifeedant activities at 48 h were Aphis gossypii G.(81.96%,87.74%)>Pieris rapae L.(75.75%,79.35%)>Plutella xylostella L.(61.20%,65.23%).(2)To investigate the effects of 70%ethanol extracts on the melanization reaction and the content of dopa,protein and blood glucose in metabolic enzymes in diamondback moth.Experiments showed that the ethanol extracts could activate the melanization reaction of hemolymph in vitro at the initial stage of administration,which was positively correlated with the concentration of the drug.However,with the change of time,the melanization reaction was inhibited.The content of dopa,the intermediate product,increased with the increase of the concentration of administration,which verified the activation of the melanization reaction of walnut leaves.At the protein level,the 70%ethanol extract of walnut leaves had an effect on the protein content in the hemolymph.With the administration of 48 h as the cut-off point,the protein content increased and then decreased,and the variation was about 10%.In the blood sugar level,the trehalose content in the hemolymph decreases with the increase of the extract concentration,and the content in the insect body first increase and then decrease with time.This is because when the insect body needs energy,the trehalose will immediately converts to glucose,which powers the body.(3)The phenoloxidase in the haemolymph of the Plutella xylostella L.was used as a target to investigate the activity of 70%ethanol extract in vitro and in vivo,and further explore the specific effects of the ethanol extract on phenoloxidase gene transcription and translation in vivo.Using L-tyrosine as substrate,the enzymatic properties of phenoloxidase from Plutella xylostella L.were studied.The results showed that the phenoloxidase of Plutella xylostella L.was stable in the range of pH = 6-6.8,the optimum pH was 6.4,and the enzyme activity was relatively stable at 20-40 ℃,the optimum temperature was 34 ℃.Experiments showed that 70%ethanol extract.had activated phenoloxidase in the Plutella xylostella L.in vitro.When the concentration was 9 mg/mL,the activation rate reached 35.09%.The phenoloxidase of Plutella xylostella L.was also activated in vivo,and the activation of the 4th instar larvae was the strongest.The activation of different tissues of the 4th instar larvae was determined and the henolymph>epidermis>head was detected.The expression of phenoloxidase mRNA and protein was explored by qRT-PCR and SDS-PAGE respectively.The results showed that the higher the concentration of the phenoloxidase was,the higher the expression level was,indicating that 70%ethanol extract at least promoted the transcriptional-level expression,and whether directly promotes the expression of translation level needs further verification. |
